Plant proteinase
inhibitors (PIs) have been well established to play a potent defensive
role against predators and pathogens. Although diverse endogenous functions
for these proteins has been proposed, ranging from regulators of endogenous
proteinases to act as storage proteins, evidence for many of these roles
is partial, or confined to isolated examples. On the other hand, many
PIs have been shown to act as defensive compounds against pests by direct
assay or by expression in transgenic crop plants, and a body of evidence
for their role in plant defense has been accumulated consistently. The
role and mechanism of action for most of these inhibitors are being
studied in detail and their respective genes isolated. These genes have
been used for the construction of transgenic crop plants to be incorporated
in integrated pest management programmes. This article describes the
classes of protease inhibitors, their regulation and genes used to construct
transgenic plants against phytophagous insects.

Article

World wide crop losses without the
use of pesticides and other non-chemical control strategies is estimated
to be about 70% of crop production, amounting to US $ 400 billion. The
world wide pre-harvest losses due to insect pests, despite, the use
of insecticides is 15% of total production representing over US $ 100
billion (Krattiger, 1997). The annual cost of insect
control itself amounts to US $ 8 billion, thus warranting urgent economical
control measures. Many of the crop varieties developed in the past 30
years were high yielders, but had poor storage characteristics (Kerin,
1994). Insect pests are capable of evolving to biotypes that can
adapt to new situations, for instance, they overcome the effect of toxic
materials or bypass natural or artificial plant resistance, which further
confounds the problem (Roush and McKenzie, 1987).
Under these circumstances, provision of food to the rapidly expanding
population has always been a challenge facing mankind. This problem
is more acute in the tropics and sub-tropics, where the climate provides
a highly condusive environment for a wide range of insects and necessitates
massive efforts to suppress the population densities of different pests
in order to achieve an adequate supply of food. In developing countries,
the problem of competition from insect pests is further complicated
with a rapid annual increase in the human population (2.5-3.0 percentage)
in comparison to a 1.0 percent increase in food production. In order
to feed the ever expanding population, crop protection plays a vital
and integral role in the modern day agricultural production to minimise
yield losses. Currently, the crop protection practice in such agricultural
systems relies exclusively on the use of agrochemicals, although a few
specific cases do exist where inherent varietal resistance and biological
control have been successfully employed. The exclusive use of chemical
pesticides not only results in rapid build-up of resistance to such
compounds, but their non-selectivity affects the balance between pests
and natural predators, and is generally in favour of pests (Metcalf,
1986). Therefore, an integrated pest management (IPM) programme,
comprising a combination of practices including the judicious use of
pesticides, crop rotation, field sanitation and above all exploitation
of inherently resistant plant varieties would provide the best option
(Meiners and Elden, 1978). The last option includes
the use of transgenic crops, expressing foreign insecticidal genes which
could make a significant contribution to sustainable agriculture and
thus could be an important component of IPM (Boulter,
1993). The production of transgenic crops has seen rapid advances
during the last decade with the commercial introduction of Bt
transgenics, but the major concern with these crops has been the development
of resistance by pest and public acceptability. Hence, there has been
a need to discover new effective plant genes which would offer resistance/protection
against these pests. Protease inhibitors (PIs) are one of the prime
candidates with highly proven inhibitory activity against insect pests
and also known to improve the nutritional quality of food.

Plant
protease inhibitors

The possible
role of protease inhibitors (PIs) in plant protection was investigated
as early as 1947 when, Mickel and Standish observed that the larvae
of certain insects were unable to develop normally on soybean products.
Subsequently the trypsin inhibitors present in soybean were shown to
be toxic to the larvae of flour beetle, Tribolium confusum (Lipke
et al. 1954). Following these early studies, there have been many
examples of protease inhibitors active against certain insect species,
both in in vitro assays against insect gut proteases (Pannetier
et al. 1997; Koiwa et al, 1998) and in in vivo
artificial diet bioassays (Urwin et al. 1997; Vain
et al. 1998).The term "protease" includes both "endopeptidases"
and "exopeptidases" whereas, the term "proteinase" is used to describe
only "endopeptidases" (Ryan, 1990). Several non-homologous
families of proteinase inhibitors are recognized among the animal, microorganisms
and plant kingdom. Majority of proteinase inhibitors studied in plant
kingdom originates from three main families namely leguminosae, solanaceae
and gramineae (Richardson, 1991).

These protease inhibitor
genes have practical advantages over genes encoding for complex pathways
i.e. by transferring single defensive gene from one plant species
to another and expressing them from their own wound inducible or constitutive
promoters thereby imparting resistance against insect pests (Boulter,
1993). This was first demonstrated by Hilder et al.
1987 by transferring trypsin inhibitor gene from Vigna unguiculata
to tobacco, which conferred resistance to wide range of insect pests
including lepidopterans, such as Heliothis and Spodoptera,
coleopterans such as Diabrotica, Anthonomnous and orthoptera
such as Locusts. Further, there is no evidence that it had toxic
or deleterious effects on mammals. Many of these protease inhibitors
are rich in cysteine and lysine, contributing to better and enhanced
nutritional quality (Ryan, 1989). Protease inhibitors
also exhibit a very broad spectrum of activity including suppression
of pathogenic nematodes like Globodera tabaccum, G. pallida,
and Meloidogyne incognita by CpTi (Williamson
and Hussey, 1996), inhibition of spore germination and mycelium
growth of Alternaria alternata by buckwheat trypsin/chymotrypsin
(Dunaevskii et al. 1997) and cysteine PIs from pearl
millet inhibit growth of many pathogenic fungi including Trichoderma
reesei (Joshi et al. 1998). These advantages make
protease inhibitors an ideal choice to be used in developing transgenic
crops resistant to insect pests. Further, transformation of plant genomes
with PI-encoding cDNA clones appears attractive not only for the control
of plant pests and pathogens, but also as a means to produce PIs, useful
in alternative systems and the use of plants as factories for the production
of heterologous proteins (Sardana et al. 1998). These
inhibitor families that have been found are specific for each of the
four mechanistic classes of proteolytic enzymes, and based on the active
amino acid in their "reaction center" (Koiwa et al.
1997), are classified as serine, cysteine, aspartic and metallo-proteases.

Serine
proteinase inhibitors

The role of
serine PIs as defensive compounds against predators is particularly
well established, since the major proteinases present in plants, used
for processes such as protein mobilization in storage tissues, contain
a cysteine residue as the catalytically active nucleophile in the enzyme
active site. Serine proteinases are not used by plants in processes
involving large scale protein digestion, and hence the presence of significant
quantities of inhibitors with specificity towards these enzymes in plants
cannot be used for the purposes of regulating endogenous proteinase
activity (Reeck et al. 1997). In contrast, a major
role for serine PIs in animals is to block the activity of endogenous
proteinases in tissues where this activity would be harmful, as in case
of pancreatic trypsin inhibitors found in mammals. The serine class
of proteinases such as trypsin, chymotrypsin and elastase, which belong
to a common protein superfamily, are responsible for the initial digestion
of proteins in the gut of most higher animals (GarciaOlmedo
et al. 1987). In vivo they are used to cleave long, essentially
intact polypeptide chains into short peptides which are then acted upon
by exopeptidases to generate amino acids, the end products of protein
digestion. These three types of digestive serine proteinases are distinguished
based on their specificity, trypsin specifically cleaving the C-terminal
to residues carrying a basic side chain (Lys, Arg), chymotrypsin showing
a preference for cleaving C-terminal to residues carrying a large hydrophobic
side chain (Phe, Tyr, Leu), and elastase showing a preference for cleaving
C-terminal to residues carrying a small neutral side chain (Ala, Gly)
(Ryan, 1990). Inhibitors of these serine proteinases
have been described in many plant species, and are universal throughout
the plant kingdom, with trypsin inhibitors being the most common type.
At least, part of this bias can be accounted for by the fact that (mammalian)
trypsin is readily available and is the easiest of all the proteinases
to assay using synthetic substrates, and hence is used in screening
procedures. Because of these reasons the members of the serine class
of proteinases have been the subject of intense research than any other
class of proteinase inhibitors. Such studies have provided a basic understanding
of the mechanism of action (Huber and Carrell, 1989)
that applies to most serine proteinase inhibitor families and probably
to the cysteine and aspartyl proteinase inhibitor families as well.
All serine inhibitor families from plants are competitive inhibitors
and all of them inhibit proteinases with a similar standard mechanism
(Laskowski and Kato, 1980).

Serine proteinases
have been identified in extracts from the digestive tracts of insects
from many families, particularly those of lepidoptera (Houseman
et al. 1989) and many of these enzymes are inhibited by proteinase
inhibitors. The order lepidoptera, which includes a number of crop pests,
the pH optima of the guts are in the alkaline range of 9-11 (Applebaum,
1985) where, serine proteinases and metallo-exopeptidases are most
active. Additionally, serine proteinase inhibitors have anti-nutritional
effects against several lepidopteran insect species (Shulke
and Murdock, 1983; Applebaum, 1985). Purified Bowman-Birk
trypsin inhibitor (Brovosky, 1986) at 5% of the diet
inhibited growth of these larvae but SBTI (Kunitz, 1945),
another inhibitor of bovine trypsin, was less effective when fed at
the same levels.

Broadway
and Duffey (1986a) compared the effects of purified SBTI and potato
inhibitor II (an inhibitor of both trypsin and chymotrypsin) on the
growth and digestive physiology of larvae of Heliothis zea and
Spodoptera exigua and demonstrated that growth of larvae was
inhibited at levels of 10% of the proteins in their diet. Trypsin inhibitors
at 10% of the diet were toxic to larvae of the Callosobruchus maculatus
(Gatehouse and Boulter, 1983) and Manduca sexta
(Shulke and Murdock, 1983).

Recent X-ray
crystallography structure of winged bean, Psophocarpus tetragonolobusKunitz-type double headed alpha-chymotrypsin shows 12 anti-parallel
beta strands joined in a form of beta trefoil with two reactive site
regions (Asn 38-Leu 43 and Gln 63-Phe 68) at the external loops (Ravichandaran
et al. 1999; Mukhopadhyay, 2000). Structural
analysis of the Indian finger millet (Eleusine coracana) bifunctional
inhibitor of alpha-amylase/trypsin with 122 amino acids has shown five
disulphide bridges and a trypsin binding loop (Gourinath
et al. 2000). These structural analysis would greatly help in "enzyme
engineering" of the native PIs to a potent form, against the target
pest species than the native PIs.

Cysteine
proteinase inhibitors

Isolation
of the midgut proteinases from the larvae of cowpea weevil, C. maculatus
(Kitch and Murdock, 1986; Campos et
al. 1989) and bruchid Zabrotes subfaceatus (Lemos
et al. 1987) confirmed the presence of cysteine mechanistic
class of proteinase inhibitors. Similar proteinases have been isolated
from midguts of the flour beetle Tribolium castaneum, Mexican
beetle Epilachnavarivestis (Murdock et
al. 1987) and the bean weevil Ascanthoscelides obtectus
(Wieman and Nielsen, 1988). Cysteine proteinases
isolated from insect larvae are inhibited by both synthetic and naturally
occurring cysteine proteinase inhibitors (Wolfson and
Murdock, 1987). In a study of the proteinases, from the midguts
of several members of the order coleopteran, 10 of 11 beetle species
representing 11 different families, had gut proteinases that were inhibited
by p-chloromercuribenzene sulfonic acid (PCMBS), a potent sulphydryl
reagent (Murdock et al. 1988) indicating that
the proteinases were of the cysteine mechanistic class. The optimum
activity of cysteine proteinases is usually in the pH range of 5-7,
which is the pH range of the insect gut that use cysteine proteinases
(Murdock et al. 1987). Another puzzling aspect of
studies with C. maculatus is the apparent effects of certain
members of Bowman-Birk trypsin inhibitor family on the growth and development
of these larvae. Although cysteine proteinase is primarily responsible
for protein digestion in C. maculatus, it is not clear, how the
cowpea and soybean Bowman-Birk inhibitors are exert their anti-nutritional
effects on this organism. Advances in enzymology has revealed the existence
of a variety of cysteine proteinases resulting in their classification
into several families namely papain, calpin and asparagines specific
processing enzyme (Turk and Bode, 1991). Cystanins
have also been characterized from potato (Waldron et
al. 1993), ragweed (Rogers et al. 1993), cowpea
(Fernandes et al. 1993) papaya (Song
et al. 1995) and avacado (Kimura et al. 1995).

The rice cysteine proteinase
inhibitors are the most studied of all the cysteine PIs which is proteinaceous
in nature (Abe and Arai, 1985) and highly heat stable
(Abe et al. 1987). Recent three dimensional structure
analysis of oryzacystatin OC-I by Tanokura's group (Nagata
et al. 2000), using NMR has showed a well defined main body consisting
of amino acids from Glu 13 - Asp 97 and an alpha helix with five stranded
anti parallel beta-sheet, while the N terminus (Ser 2-Val 12) and C
terminus (Ala 98-Ala 102) are less defined. Further, analysis has demonstrated
OC-I to be similar to chicken cystatin which belongs to type-2 animal
cystatin. Another rice cystatin named as OC-II, with a putative target
binding motif gln-x-val-x-gly shares similar motif with OC-I but has
a different inhibition constant (Ki) value (Arai et al.
1991; Kondo et al. 1991).

Aspartic
and metallo-proteinase inhibitors

Knowledge
on the role of aspartic proteinases in insect digestion is limited than
that of cysteine proteinases. In species of six families of the order
hemiptera, aspartic proteinases (cathepsin D-like proteinases) were
found along with cysteine proteinases (Houseman and Downe,
1983). The low pH of midguts of many members of coleoptera and hemiptera
provides more favourable environments for aspartic proteinases (pH optima
~ 3-5) than the high pH of most insect guts (pH optima ~ 8-11) (Houseman
et al. 1987) where the aspartic and cysteine proteinases
would not be active. No aspartic proteinases have been isolated from
coleoptera but Wolfson and Murdock 1987 demonstrated
that pepstatin, a powerful and specific inhibitor of aspartyl proteinases,
strongly inhibited proteolysis of the midgut enzymes of Colorado potato
beetle, Leptinotarsa decemlineata indicating that an aspartic
proteinase was present in the midgut extracts. Potato tubers possess
an aspartic proteinase inhibitor, cathepsin D (Mares et
al. 1989) that shares considerable amino acid sequence identity
with the trypsin inhibitor SBTI from soybeans. Plants have also evolved
at least two families of metallo-proteinase inhibitors, the metallo-carboxypeptidase
inhibitor family in potato (Rancour and Ryan, 1968),
and tomato plants (Graham and Ryan, 1981) and a cathepsin
D inhibitor family in potatoes (Keilova and Tomasek, 1976).

The cathepsin D inhibitor
(27 kDa) is unusual as it inhibits trypsin and chymotrypsin as well
as cathepsin D, but does not inhibit aspartyl proteases such as pepsin,
rennin or cathepsin E. The inhibitors of the metallo-carboxypeptidase
from tissue of tomato and potato are polypeptides (4 kDa) that strongly
and competitively inhibit a broad spectrum of carboxypeptidases from
both animals and microorganisms, but not the serine carboxypeptidases
from yeast and plants (Havkioja and Neuvonen, 1985).
The inhibitor is found in tissues of potato tubers where it accumulates
during tuber development along with potato inhibitor I and II families
of serine proteinase inhibitor. The inhibitor also accumulates in potato
leaf tissues along with inhibitor I and II proteins in response to wounding
(Graham and Ryan, 1981; Hollander-Czytko
et al. 1985). Thus, the inhibitors accumulated in the wounded leaf
tissues of potato have the capacity to inhibit all the five major digestive
enzymes i.e. trypsin, chymotrypsin, elastase, carboxypeptidase
A and carboxypeptidase B of higher animals and many insects (Hollander-Czytko
et al. 1985). Aspartic PIs have been recently been isolated from
sunflower (Park et al. 2000), barley (Kervinen
et al. 1999) and cardoon (Cyanara cardunculus) flowers named
as cardosin A (Frazao et al. 1999).

The detailed structural
analysis of prophytepsin, a zymogen of barley aspartic proteinase shows
a pepsin like bilobe and a plant specific domain. The N terminal has
13 amino acids necessary for inactivation of the mature phytepsin (Kervinen
et al. 1999), and the aspartic PI cardosin A from cardoon shows
regions of glycolylations at Asn-67 and Asn 257. The Arg-Gly-Asp sequences
recogonizes the cardosin receptor which is found in a loop between two-beta
strands on the molecular surface (Frazao et al. 1999).

Mechanism
of toxicity

The mechanism
of action of these proteinase inhibitors has been a subject of intense
investigation (Barrett, 1986; MacPhalen
and James, 1987; Greenblatt et al. 1989).
Knowledge on mechanisms of protease action and their regulation in
vitro, and in vivo, in animals, plants, microorganisms and
more recently in viruses have contributed to many practical applications
for inhibitor proteins in medicine and agriculture.

Baker
et al. 1984 showed that the secretion of proteases in insect
guts depends upon the midgut protein content rather than the food volume.
The secretion of proteases has been attributed to two mechanisms, involving
either a direct effect of food components (proteins) on the midgut epithelial
cells, or a hormonal effect triggered by food consumption (Applebaum,
1985). Models for the synthesis and release of proteolytic enzymes
in the midguts of insects proposed by Birk and Applebaum,
1960, Brovosky, 1986 reveal that ingested food
proteins trigger the synthesis and release of enzymes from the posterior
midgut epithelial cells. The enzymes are released from membrane associated
forms and sequestered in vesicles that are in turn associated with the
cytoskeleton. The peptidases are secreted into the ectoperitrophic space
between the epithelium, as a particulate complex (Eguchi
et al. 1982), from where the proteases move transversely
into the lumen of the gut, where the food proteins are degraded. PIs
inhibit the protease activity of these enzymes and reduce the quantity
of proteins that can be digested, and also cause hyper-production of
the digestive enzymes which enhances the loss of sulfur amino acids
(Shulke and Murdock, 1983) as a result of which,
the insects become weak with stunted growth and ultimately die.

The digestive
proteolytic enzymes in the different orders of commercially important
insect pests belong to one of the major classes of proteinases predominantly.
Coleopteran and hemipteran species tend to utilize cysteine proteinases
(Murdock et al. 1987) while lepidopteran, hymenopteran,
orthopteran and dipteran species mainly use serine proteinases (Ryan,
1990; Wolfson and Murdock, 1990). Examples from
both of these classes of proteinases have been shown to be inhibited
by their cognate proregions (Taylor et al. 1995).
The effect of class specific inhibitors on the pest digestive enzymes
is not always a simple inhibition of proteolytic activity, but recent
studies have indicated the reverse may happen. It would appear that
there are often two populations of digestive enzymes in target pests,
those that are susceptible to inhibition and those that are resistant
(Michaud et al. 1996; Bown et al. 1997),
and some insects respond to ingestion of plant PIs such as soybean trypsin
inhibitor (Broadway and Duffey, 1986b) and oryzacystatin
(Michaud et al. 1996) by hyper-producing inhibitor-resistant
enzymes.

The mechanism
of binding of the plant protease inhibitors to the insect proteases
appear to be similar with all the four classes of inhibitors. The inhibitor
binds to the active site on the enzyme to form a complex with a very
low dissociation constant (107 to 1014 M at neutral
pH values), thus effectively blocking the active site. A binding loop
on the inhibitor, usually "locked" into conformation by a
disulphide bond, projects from the surface of the molecule and contains
a peptide bond (reactive site) cleavable by the enzyme (Terra
et al. 1996; Walker et al. 1998). This peptide
bond may be cleaved in the enzyme inhibitor complex, but cleavage does
not affect the interaction, so that a hydrolyzed inhibitor molecule
is bound similar to an unhydrolyzed one. The inhibitor thus directly
mimics a normal substrate for the enzyme, but does not allow the normal
enzyme mechanism of peptide bond cleavage to proceed to completion ie.,
dissociation of the product (Walker et al. 1998).
It would also appear that insect digestive trypsins do not fall into
the classification of peptidase hydrolases, as defined by inhibition
spectra. It has been shown, notably, that the trypsin like digestive
proteases of several lepidopteran species are inhibited by (l-3-trans
carboxiran-2-carbonyl)-l-leu-agmatin (E-64) (Lee and Anstee,
1995; Novillo et al. 1997) an inhibitor generally
considered to be specific to cysteine proteinases (Dunn,
1989). Thus, true interactions will become clear only when we have
protein crystals and X-ray diffraction data for the structure of insect
enzyme/inhibitor complexes. Further, specificity of the inhibitor enzyme
interaction is primarily determined by the specificity of proteolysis
determined by the enzyme (Blancolabra et al. 1996).

Regulation
of proteinase inhibitors

Plant proteinase
inhibitor proteins that are known to accumulate in response to wounding
have been well characterized. Earlier research on tomato inhibitors
has shown that the protease inhibitor initiation factor (PIIF), triggered
by wounding/injury switches on the cascade of events leading to the
synthesis of these inhibitor proteins (Melville and Ryan,
1972; Bryant et al. 1976), and the newly
synthesized PIs are primarily cytosolic (Hobday et al.
1973; Meige et al. 1976).

The current
evidence suggests that the production of the inhibitors occurs via.
the octadecanoid (OD) pathway, which catalyzes the break down of linolenic
acid and the formation of jasmonic acid (JA) to induce protease inhibitor
gene expression (Koiwa et al. 1997). There
are four systemic signals responsible for the translocation of the wound
response, which includes systemin, abscisic acid (ABA), hydraulic signals
(variation potentials) and electrical signals (Malone
and Alarcon, 1995). These signal molecules are translocated from
the wound site through the xylem or phloem as a consequence of hydraulic
dispersal. The plant systemin an 18-mer peptide has been intensely studied
from wounded tomato leaves which strongly induced expression of protease
inhibitor (PI) genes. Transgenic plants expressing prosystemin antisense
cDNA exhibited a substantial reduction in systemic induction of PI synthesis,
and reduced capacity to resist insect attack (McGurl et
al. 1994). Systemin regulates the activation of over 20 defensive
genes in tomato plants in response to herbivorous and pathogenic attacks.
The polypeptide activates a lipid-based signal transduction pathway
in which linolenic acid, is released from plant membranes and converted
into an oxylipin signaling molecule, jasmonic acid (Ryan,
2000). A wound-inducible systemin cell surface receptor with an
M(r) of 160,000 has also been identified and the receptor regulates
an intracellular cascade including, depolarization of the plasma membrane
and the opening of ion channels thereby increasing the intracellular
Ca(2+), which activates a MAP kinase activity and a phospholipase A(2).
These rapid changes, play a vital role leading to the intracellular
release of linolenic acid from membranes and its subsequent conversion
to JA, a potent activator of defense gene transcription (Ryan,
2000). The oligosaccharides, generated from the pathogen-derived
pectin degrading enzymes i.e. polygalacturonase (Bergey
et al. 1999) and the application of systemin as well as wounding
have been shown to increase the jasmonate levels in tomato plants. Application
of jasmonate or its methyl ester, methyl jasmonate, strongly induces
local and systemic expression of PI genes in many plant species, suggesting
that jasmonate has an ubiquitous role in the wound response (Wasternack
and Parthier, 1997). Further, analysis of a potato PI-IIK promoter
has revealed a G-box sequence (CACGTGG) as jasmonate-responsive element
(Koiwa et al. 1997). The model developed for the wound-induced
activation of the proteinase inhibitor II (Pin2) gene in potato (Solanum
tuberosum) and tomato (Lycopersicon esculentum) establishes
the involvement of the plant hormones, abscisic acid and jasmonic acid
(JA) as the key components of wound signal transduction pathway (Titarenko
et al. 1997). Recently, it has been shown that the defense signaling
in suspensions of cultured cells of Lycopersicon peruvianum by
peptide systemin, chitosan and by beta-glucan elicitor from Phytophtora
megasperma, is inhibited by the polysulfonated naphtyl urea compound
suramin, a known inhibitor of cytokine and growth factor receptor interactions
in animal cells (Stratmann et al. 2000a). Levels
of ABA have been shown to increase in response to wounding, electrical
signal, heat treatment or systemin application in parallel with PI induction
(Koiwa et al. 1997). Abscisic acid originally
thought to be involved in the signaling pathway is now believed to weakly
induce the mRNAs of wound response proteins and a concentration even
as high as 100 mM induced only low levels of proteinase inhibitor as
compared to systemin or jasmonic acid (Birkenmeiner and
Ryan, 1998), suggesting the localized role of ABA.

However, it
is evident that wound induction and pathogen defense pathways overlap
considerably. Expression of wound and JA inducible genes can be positively
and negatively regulated by ethylene or salicylic acid (SA), both of
which are components of the pathogen-induced signaling pathway (Bent,
1996; Delaney et al. 1994). The expression of
thionins in Arabidopsis (Epple et al. 1995)
and lectin II in Griffonia simplicifolia (Zhu-Salzman
et al. 1998) was elicited by JA but suppressed by ethylene,
showing their opposite effects on the wound signaling pathway.

Plants sometimes specifically
forego one type of defense response for another. Salicylic acid (SA)
and its methyl ester (Me-SA) are both defense compounds that potently
induce systemic acquired resistance of plants against pathogenic microorganisms
(Hunt et al. 1996). However, in response to spider
mite infestation, lima bean plants release Me-SA which functions as
a volatile attractant of the predatory mite Amblyseius potentillae
(Dicke et al. 1990). At the same time, SA itself negatively
regulates the OD pathway through inhibition of SA biosynthesis and activity
(Korth and Dixon, 1997), indicating that SA may suppress
the plant defense response through attenuation of the OD pathway, but
its methyl ester positively affects plant defense through another defense
mechanism involving tritrophic plant herbivore interaction (Moura
and Ryan, 2001). Different jasmonic acid-dependent and independent
wound signal transduction pathways have been identified recently and
partially characterized. Components of these signalling pathways are
mostly similar to those implicated in other signalling cascades which
include reversible protein phosphorylation steps, calcium/calmodulin-regulated
events, and production of active oxygen species (León
et al. 2001).

Stintzi
et al. (2001) using biochemical genetic approach demonstrated
that cyclopentenone precursor of JA, 12-oxo-phytodienoic acid (OPDA),
as a physiological signal eliciting defensive response and resistance
in the absence of JA. Studies on the effect of UV radiation on early
signaling events in the response of young tomato plants to ultraviolet-C
(< 280 nm) and UVB/UVA (280-390 nm) radiation induces a 48 kDa myelin
basic protein kinase activity in leaves (Stratmann et
al. 2000b). In the case of barley plants, ethylene increased the
activity of both cell wall bound peroxidases types (ionically and covalently
bound), comparable with infestation, which suggests that ethylene is
involved in the oxidative responses of (Argandoña et al.
2001)

Studies
on the induction of PI proteins have indicated a de novo synthesis
of proteins such as a Boman Birk protease inhibitor (OsBBPI) from rice
which was found to be rapidly induced in seedling leaf in response to
cut, exogenous jasmonic acid (JA), and two potent protein phosphatase
2A (PP2A) inhibitors, in a light/dark, time and dose dependent manner
but was completely inhibited by cycloheximide (Rakwal
et al. 2001).

Structure
of protease inhibitor genes

The gene size
and coding regions of the inhibitors are generally small with no introns
(Boulter, 1993) and many of these inhibitors are products
of multigene families (Ryan, 1990). Bowman-Birk type
double-headed protease inhibitors are assumed to have arisen by duplication
of an ancestral single headed inhibitor gene and subsequently diverged
into different classes i.e. trypsin/trypsin (T/T), trypsin/chymotrypsin
(T/C) and trypsin/elastase (T/E) inhibitors (Odani et
al. 1983). The mature proteins comprise a readily identifiable 'core'
region, covering the invariant cysteine residues and active center serines,
which are bound by highly variable amino and carboxy-terminal regions.
There is a core region of 62 amino acids, both between and within the
different classes of inhibitor, within cowpea and with other leguminosae,
including azuki bean (Ishikawa et al. 1985), lima
bean (Stevens et al. 1974), mung bean (Zhang
et al. 1982) and soybean (Odani and Ikenaka,
1976). The average number of amino acid replacements in this region
from all pair-wise comparisons show that the differences between the
different classes of inhibitor within a species (around 16.5/62 residues)
are much greater than the differences within a class between different
species (around 11/62 residues). Considering that 18 of the residues
in this region are obligatorily invariant for proteins to be classified
as Bowman-Birk type inhibitors, these are very high rates of amino acid
substitutions. This highlights the problems likely to be encountered
in attempting to draw conclusions about the evolutionary history of
the rapidly diverging, multigenic protein families from sequences which
may be paralogous rather than orthologous. Corrected divergence between
pair-wise combinations of sequences calculated according to the method
of Perler et al. 1980 revealed that the average divergence
between trypsin-specific and chymotrypsin-specific second domains (about
36%) is very similar to that between the first and second domains (about
40%). On an "evolutionary clock" model this would imply that the gene
duplication leading to T/T and T/C families occurred very close to the
duplication, leading to the appearance of the double-headed inhibitors
and that the number of silent substitutions has reached saturation in
all these genes (Hilder et al. 1989).

Analysis of the winged
bean Kunitz chymotrypsin inhibitor (WCI) protein shows that it is encoded
by a multigene family that includes four putative inhibitor coding genes
and three pseudogenes. The structural analysis of the WCI genes indicates
that an insertion at a 5' proximal site occurred after duplication of
the ancestral WCI gene and that several gene conversion events subsequently
contributed to the evolution of this gene family (Habu
et al. 1997). The 5' region of the pseudogene, WCI-P1 contains frameshift
mutations, an indication that the 5' region of the WCI-P1 gene may have
spontaneously acquired new regulatory sequences during evolution. Since,
gene conversion is a relatively frequent event and the homology between
the WCI-P1 and the other inhibitor genes WCI-3a/b is disrupted at a
5' proximal site by remnants of an inserted sequence, the WCI-P1 gene
appears to be a possible intermediate that could be converted into a
new functional gene with a distinct pattern of expression by a single
gene-conversion event (Habu et al. 1997). Molecular
evolution of wip-1 genes from four Zea species show significant
heterogeneity in the evolutionary rates of the two inhibitory loops,
in which one inhibitory loop is highly conserved, whereas the second
is diverged rapidly. Because these two inhibitory loops are predicted
to have very similar biochemical functions, the significantly different
evolutionary histories suggest that these loops have different ecological
functions (Tiffin and Gaut, 2001).

The 3'ends of the sequences
are comprised of alternating purine-rich and pyrimidine-rich traits
of nucleotide. The first of these purine-rich trait occurs at the C-terminal
region of the coding sequence. Mutations within this region (deletion/additions
or base changes) give rise to substitution amongst the A/G rich codons,
Asp, Glu, Lys, Asn and termination codons and also certain specific
motifs within them appear to be relatively conserved, therefore likely
to be of functional significance. This obviously applies to the cononical
polyadenylation signals in the third and second purine-rich traits and
the 3'region has regulatory elements which are dependent on a higher
order of structure than the specific base sequence. X-ray crystallography
of Bowman-Birk inhibitors suggests that this termini has no role in
the interaction of the inhibitor with its target enzyme (Suzuki
et al. 1987). Substitutions and deletion/additions appear to be
very feasible in this region, provided that there is a cleavable serine
or asparagine residue within 10-20 amino acids of the first cysteine
(Ryan, 1990). The inhibitors are synthesized as precursors
from which the leader sequence is cleaved and a long trait of leader-encoding
sequence is present in soybean genomic clones (Hilder
et al. 1989). There is no significant homology in this region to
other seed-expressed protein leader sequences, other than a high representation
of hydrophobic residues. Multiple potential initiator codons are a common
feature of legume seed protein genes exemplifying the high degree of
evolutionary novelty which appears to be tolerated within such seed
specific secondary compound genes (Hilder et al. 1989).
Analysis of oryzacystatin OC-I has revealed the presence of two introns;
the first a 1.4 kbp region between Ala 38 and Asn 39 and a second region
of 372 bp in the 3' non coding region (Kishimoto et al.
1994) and a second oryzacystatin, OC-II present on chromosome 5
also has introns in the same positions (Kondo et al. 1991)
thus suggesting deviation from the earlier PIs which lacked introns.

Developing
insect resistant transgenic plants expressing Pls

A large number
of protease inhibitor genes with distinct modes of action have been
isolated from a wide range of crop species. Development of transgenic
crops have come a long way from the first transgenic developed by Hilder
et al. 1987. Considering the high complexity of protease/inhibitor
interactions in host pest systems and the diversity of proteolytic enzymes
used by pests and pathogens to hydrolyze dietary proteins or to cleave
peptide bonds in more specific processes (Graham et al.
1997), the choice of an appropriate proteinase inhibitor (PI) or
set of PIs represents a primary determinant in the success or failure
of any pest control strategy relying on protease inhibition. Firstly,
the choice of suitable PIs should be based on a detailed understanding
of the biological system assessed. Based on our current knowledge about
the use of specific inhibitors in the study and control of various metabolic
pathways, many PIs have been used to create transgenic crop plants as
shown in Table 1 and many more inhibitors are
also being isolated with divergent modes of action against different
pest species (Table 2). Resistant biotypes of
insects may evolve after prolonged exposure to selection pressure that
is mediated by an insecticidal protein or plant resistance gene (Sparber,
1985). Unless the biotechnology strategy is designed and implemented
to overcome these problems, it will become ineffective in due course
like any pesticide based management strategy.

Second point
to consider would be the targeted expession of PIs in response to pest
attack. This will be controlled by using inducible promoters, such as
those of PI-II85 and TobRB7, that are activated at the site of invasion
by pests, pathogen and nematodes, respectively (Opperman
et al.1994). An ideal promoter should be highly responsive to invasion
of the host plant by a pest, or regulated by inducers just prior to
pest attack. The promoter should be sufficiently active to mediate a
substantial defense, specially localized to the site of pest invasion.
Suitable promoters such as those regulated in response to pest invasion
can be identified using promoter trapping techniques (Babiychuk
et al. 1997).

Despite these promising
developments, the general usefulness of recombinant PIs in plant protection
still remains to be demonstrated. The inhibitory spectrum of PIs is
usually limited to proteases in one of several mechanistic classes,
leaving free proteases in the surrounding medium after inhibition (Barrett,
1994). Due to a progressive adaptation of plant pests to the continuous
occurrence of PIs in the diet, the inhibitory spectrum of protein inhibitors
against the extracellular proteases of several pests is even more limited,
being often restricted to the family level (Michaud et
al. 1995b; Visal et al. 1998). Non-target proteases,
that may allow metabolic compensation of inhibited proteolytic functions,
(Jongsma and Bolter, 1997) may also challenge the
structural integrity of several PIs and thus potentially affect their
effectiveness in vivo (Michaud, 1997).

Recently it has been
shown that the presence of large amounts of inhibitors including soybean
Kunitz inhibitor (Bown et al.1997) in the diets of
economical pests has made insects to adapt and produce proteases which
are insensitive to the action of host plant inhibitors and the ingested
PIs activate these genes (Dennis et al. 1994). As
a result, pest control using PIs in transgenic plants requires the isolation
of inhibitors that are active towards these insensitive proteases (Jongsma
et al.1996). One can search for active inhibitors among naturally-occurring
peptides (Gruden et al. 1998) or can engineer inhibitors
in such a way that they will acquire activity against the "PI insensitive"
protease. Engineering of inhibitors can be performed in two distinct
ways: 1) based on structures of the inhibitor-protease complex, predictions
can be made on mutations that will enhance binding (Urwin
et al. 1995), but lack of data on these complexes for insect proteases
makes this procedure rather tough. However, it may be more appropriate
to simply generate large arrays of mutants in the region of the inhibitor
protein contacting the protease. The powerful method of phage display
can subsequently be used to select the strongly binding mutants. Large
number of PIs have been subjected to phage display as a result of which
inhibition constant (Ki) for target protease which was initially poor
(millimolar to micromolar) have been greatly improved. Some of the examples
belonging to serine class are serpin (Pannekoek et al.
1993) and kazal from human (Rottgen and Collins,
1995), E.coli ecotin (Wang et al. 1995),
proteinase inhibitor II (PI-II) from potato (Jongsma et
al. 1995), chicken cystanin (Tanaka et al. 1995)
and soybean phytocystanin (Koiwa et al. 1998) belonging
to cysteine class. The improvement of plant PIs by phage display is
still an infant stage to be commercially important.

Insect midgut contains
an estimated 1020 different proteases (Bown et al. 1997)
which are differentially regulated and all cannot be inhibited by plant's
PIs (Broadway, 1997). Therefore, to achieve an effective
pest control strategy it is very important to achieve different inhibitors
expression in a concerted manner.

Concluding
Remarks

The availability
of diverse genes from different plant species makes it a possibility
to use one or more genes in combination, whose products are targeted
at different biochemical and physiological processes within the insect.
These packages will not only contain protease inhibitor genes but also
lectins, alpha-amylase inhibitors, or other plant genes encoding insecticidal
proteins. This technology may not replace the use of chemical pesticides
in near future but effectively complement it. The use of recombinant
PIs may also be an attractive way to protect plants from fungal, bacterial
and viral pathogens. Currently, two principal strategies are proposed
to engineer effective pest control in plants: ectopic expression of
pesticidal proteins, and induction of the plant natural defensive response.
At present, screening gene pools without taxonomic constraint can help
identify novel insecticidal determinants, but in future this approach
will be augmented by directed in vitro molecular evolution (Koiwa
et al. 1998). Given the number of pesticidal proteins that are involved
in host plant defense, it is presumed that effective pest control by
this strategy will result from the co-expression of numerous determinants,
each of which could be custom engineered by directed molecular evolution
to maximize its effectiveness against specific pests.

However, in future non-scientific
issues such as regulatory approval, propriety rights and public perception
will be decisive in releasing crop plants produced by genetic engineering
using recombinant DNA technology.

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